Preparation of hymenialdsine derivatives and use thereof

ABSTRACT

The synthesis and biological activity of indoloazepines and acid amine salts thereof which are structurally related to naturally-occurring hymenialdisine is disclosed. Naturally-occurring hymenialdisine obtained from the sponge is a potent inhibitor of production of cytokines interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). The chemically-synthesized indoloazepines of the invention also inhibit production of IL-2 and TNF-α. The indoloazepines are useful for treating inflammatory diseases, particularly diseases associated with kinases NF-κB or GSK-3β activation or NF-κB activated gene expression products. The indoloazepines are useful for the treatment of cancer by the inhibition of kinases CHK1 and CHK2.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of copending application Ser. No.10/833,871 filed on Apr. 28, 2004, which claims the benefit of U.S.Provisional Application No. 60/471,671 filed May 19, 2003.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

Reference to a “Computer Listing Appendix submitted on Disc”

Enclosed is Computer Listing Appendix submitted on disc.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention related to the synthesis and biological activityof indoloazepines and acid amine salts thereof which are structurallyrelated to naturally-occurring hymenialdisine. Thechemically-synthesized indoloazepines inhibit production of IL-2 andTNF-α. Exposure of the chemically-synthesized indoloazepine to mammalianJurkat leukemia T-cells and THP-1 cells results in a dose responseinhibition of IL-2 production and TNF-α production, respectively. Theindoloazepines are useful for treating inflammatory diseases,particularly diseases associated with NF-κB or GSK-3β activation andNF-κB activated gene expression.

(2) Description of Related Art

Elevated levels of cytokines, such as interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), andtumor necrosis factor-α (TNF-α) have been linked to many inflammatorydisorders including Crohn's disease and psoriasis, and play an essentialrole in the pathogenesis of rheumatoid arthritis and osteoarthritis(Gerard et al., Nat. Immunol. 2: 108-115 (2001); Inoue et al., Inflamm.Res. 50: 65-72 (2001); Polisson, Curr. Rheumatol. Rep. 3: 489-495(2001); Miossec, Cell Mol. Biol. (Noisy-le-grand) 47: 675-678 (2001);Roshak et al., J. Pharmacol. Exp. Ther. 283: 955-961 (1997); Harris, N.Engl. J. Med. 322: 1277-1289 (1990)).

Using anti-inflammatory drugs to inhibit cytokines, in particular TNF-α,has been successful in several clinical trials for treating rheumatoidarthritis (Moreland, J. Rheumatol. 26 Suppl. 57: 7-15 1999); Moreland,et al., N. Engl. J. Med. 337: 141-147 (1997)). However, there isvariability in responses to these anti-inflammatory drugs because of thecomplex network of alternative cytokine mediated pathways (Miossec, CellMol. Biol. (Noisy-le-grand) 47: 675-678 (2001); Handel et al., Clin.Exp. Pharmacol. Physiol. 27: 139-144 (2000)). Using drugs which inhibittranscription factors that control the expression of severalpro-inflammatory mediators, such as the nuclear transcription factorNF-κB, may overcome response variability and may provide an alternativestrategy for treating a wide variety of inflammatory disorders (Makarov,Arthritis Res. 3: 200-206 (2001); Tak, J. Clin. Invest. 107: 7-11(2001); Roshak et al., Curr. Opin. Pharmacol. 2: 316-321 (2002);Yamamoto and Gaynor, J. Clin. Invest. 107: 135-142 (2001); Feldmann etal., Ann. Rheum. Dis. 61 Suppl. 2: ii13-18 (2002); Barnes and Karin, N.Engl. J. Med. 336: 1066-1071 (1997); Lee and Burckart, J. Clin.Pharmacol. 38: 981-993 (1998); Baldwin, Ann. Rev. Immunol. 14: 649-683(1996); Miagkov et al., Proc. Natl. Acad. Sci. USA 95: 13859-13864(1998); Guttridge et al., Mol. Cell Biol. 19: 5785-5799 (1999); Baeuerleand Henkel, Ann. Rev. Immunol. 12: 141-179 (1994)).

Because of its critical role in the regulation of inflammatoryresponses, NF-κB has become an increasingly significant therapeutictarget for controlling diseases such as asthma, rheumatoid arthritis,multiple sclerosis, and Alzheimer's disease (Tak, J. Clin. Invest. 107:7-11 (2001); Yamamoto and Gaynor, J. Clin. Invest. 107: 135-142 (2001);Barnes and Karin, N. Engl. J. Med. 336: 1066-1071 (1997); Boland,Biochem. Soc. Trans. 29: 674-678 (2001); Hart et al., Am. J. Respir.Crit. Care Med. 158: 1585-1592 (1998); Yamamoto and Gaynor, Curr. Mol.Med. 1: 287-296 (2001)).

Hymenialdisine is a bromopyrrole alkaloid which was originally isolatedfrom the marine sponges Axinella verrucosa and Acantella aurantiaca. Itsstructure was established on the basis of X-ray crystallography (Ciminoet al., Tet. Lett. 23: 767-768 (1982)). The structure of hymenialdisineis shown in FIG. 1A. Hymenialdisine has been found to inhibit variousproinflammatory cytokines, such as IL-1, IL-6, IL-8, and nitric oxide ina variety of cell lines (Inoue et al., Inflamm. Res. 50: 65-72 (2001);Roshak et al., J. Pharmacol. Exp. Ther. 283: 955-961 (1997); Breton andChabot-Fletcher, J. Pharmacol. Exp. Ther. 282: 459-466 (1997); Badger etal., J. Pharmacol. Exp. Ther. 290: 587-593 (1999)). Investigation of thepromising anti-inflammatory properties of hymenialdisine revealed thatit inhibits cytokine production by inhibiting the NF-κB signalingpathway (Roshak et al., J. Pharmacol. Exp. Ther. 283: 955-961 (1997);Breton and Chabot-Fletcher, J. Pharmacol. Exp. Ther. 282: 459-466(1997); Badger et al., J. Pharmacol. Exp. Ther. 290: 587-593 (1999);Badger et al., Osteoarthritis Cartilage 8: 434-443 (2000)). Gel shiftanalysis showed that this inhibition was that hymenialdisine selectivelyreduced NF-κB nuclear binding and not the binding of other transcriptionfactors such as C/EBP, AP-1, or SP1 (Roshak et al., J. Pharmacol. Exp.Ther. 283: 955-961 (1997)).

Recently, Meijer et al. reported that the potent NF-κB inhibitorhymenialdisine acts as a competitive nanomolar inhibitor of thecyclin-dependent kinases GSK-3β and CK1 (Meijer et al., Chem. Biol. 7:51-63 (2000)). Crystallographic data showed that hymenialdisine binds tothe ATP binding pocket of the GSK-3β and CK1 kinases (Meijer et al.,Chem. Biol. 7: 51-63 (2000)). Considering the potential relationshipbetween NF-κB activation and GSK-3, that might suggest a potentialpathway for this kinase inhibitor (Meijer et al., Chem. Biol. 7: 51-63(2000); Ali et al., Chem. Rev. 101: 2527-2540 (2001); Schwabe andBrenner, Am. J. Physiol. Gastrointest. Liver Physiol. 283: G204-211(2002)). In addition, Ireland et al. identified hymenialdisine as a verypotent MEK-1 inhibitor with low nanomolar IC₅₀ values. This suggeststhat hymenialdisine may be useful as an antiproliferative agent(Tasdemir et al., J. Med. Chem. 45: 529-532 (2002)).

Hymenialdisine and various derivatives thereof such asdebromohymenialdisine have been disclosed in the following patents andpublished patent applications.

U.S. Pat. No. 5,565,448 to Nambi et al. discloses medicants whichcontain hymenialdisine or debromohymenialdisine and which are used toinhibit protein kinase C and U.S. Pat. No. 5,616,577 to Nambi et al.discloses methods using hymenialdisine or debromohymenialdisine toinhibit protein kinase C.

U.S. Pat. No. 5,591,740 to Chipman et al. discloses using compositionscomprising hymenialdisine or debromohymenialdisine to treatosteoarthritis.

U.S. Pat. No. 5,621,099 to Annoura et al. discloses a method forsynthesizing hymenialdisine, bromohymenialdisine, and related compounds.

U.S. Pat. Nos. 5,834,609, 6,103,899, and 6,218,549, all to Horne et al.disclose bicyclic aminoimidizole compounds which have anti-tumor andantimicrobial activity.

U.S. Pat. Nos. 6,197,954 B1, 6,211,361, 6,528,646, and published U.S.Patent Application No. 2001/0012891A1, all to Horne et al., discloseprocesses for synthesizing hymenialdisine, related compounds, and theirintermediates.

Published U.S. Patent Application No. 20030060457A1 to Schaffer et al.discloses that hymenialdisine is a cdk inhibitor which can be used as aninhibitor of gene expression, replication, and reactivation inpathogenic agents.

EP1106180A1 and WO0141768A2 to Meijer disclose using hymenialdisine andrelated compounds such as debromohymenialdisine to inhibit cyclindependent kinases, GSK-3β, and casein kinase 1 for preventing andtreating neurodegenerative disorders such as Alzheimer's disease,diabetes, inflammatory pathologies, and cancers.

DNA replication is a process that requires great accuracy and relies onsurveillance mechanisms, which monitor DNA damage and initiate DNArepair (Zhou, B.-B. S., et al., Nature 408 433-439 (2000)). Theinability to carry out DNA repair often leads to the transformation ofnormal cells into malignancies (Martin, N. M. B., J. Photochem.Photobiol. B 63 162-170 (2001)). Upon DNA damage, cell cycle checkpointsget activated, which delay cell cycle progression and allow DNA repair.A multi-faceted involvement of these checkpoint pathways regulates DNArepair, (Zhou, B.-B. S., et al., Nature 408 433-439 (2000; Martin, N. M.B., J. Photochem. Photobiol. B 63 162-170 (2001); and Zhao, S., et al.,Nature 405, 473 (2000)) telomere length (Naito, T., et al., Nat. Genet.20 203-206 (1998); Ritchie, K. B., et al., Mol. Cell Biol. 10 6065-6075(1999)) and the induction of apoptotic cell death (Zhou, B.-B. S., etal., Nature 408 433-439 (2000); and Lowe, S. W., et al., Nature 362847-849 (1993)). Protein kinases regulate a host of cellular processessuch as growth and differentiation, cell proliferation and apoptosis(Sielecki, T. M., et al., J. Med. Chem. 43 1-18 (2000); Sridhar, R., etal., Pharm. Res. 17 1345-1353 (2000); Traxler, P., et al., J. Med. Res.Rev. 21 499-512 (2001); Scapin, G., Drug Discovery Today 7. (2001);Toogood, P. L., Med. Res. Rev. 21 487-498 ((2001); and Bridges, A. J.,Chem. Rev. 101 2541-2572 (2001)). DNA damage caused by radiation orchemotherapy triggers the DNA damage-responsive protein kinases ATM andATR, which activate Chk1 and Chk2. Chk1 and Chk2 in turn phosphorylateCdc25 and prevent Cdc2 activation, resulting in cell cycle arrest(Curman, D., et al., J. Biol. Chem. 276 17914-17919 (2001)). Hence,small molecules that can inhibit the checkpoints may enhance theefficacy of DNA damaging chemotherapeutics or radiation therapy (Rundle,N. T., et al., J. Biol. Chem. 276 48231-48236 (2001); Jackson, J. R., etal., Cancer Res. 60 566-572 (2000); Koniaras, K., et al., Oncogene 207453-7463 (2001); Zhou, B.-B. S., et al., Cancer Biol. Ther. 2 S16-S22(2003); Yu, Q., et al., Cancer Res. 62 5743-5748 (2002)).

In light of the prior art, there remains a need for other smallmolecules that have activities which exhibit a similar or betterpharmacological profile to hymenialdisine and which are simple andinexpensive to prepare.

SUMMARY OF THE INVENTION

The synthesis and biological activity of indoloazepines and acid aminesalts thereof which are structurally related to naturally-occurringhymenialdisine is disclosed. Naturally-occurring hymenialdisine obtainedfrom the sponge is a potent inhibitor of production of interleukin-2(IL-2) (IC₅₀=2.4 μM) and tumor necrosis factor-α (TNF-α) (IC₅₀=1.4 μM).The chemically-synthesized indoloazepine also inhibits production ofIL-2 and TNF-α. Exposure of the chemically-synthesized indoloazepine tomammalian Jurkat leukemia T-cells and THP-1 cells resulted in a doseresponse inhibition of IL-2 production (IC₅₀=3.5 μM) and TNF-αproduction (IC₅₀=8.2 μM), respectively. The indoloazepines are usefulfor treating inflammatory diseases, particularly diseases associatedwith NF-κB activation.

The chemically-synthesized indoloazepine also inhibits the kinase CHK2(IC₅₀=8 nM. The indolozaepines are useful for treating cancer.

Therefore, the present invention provides a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits kinases or NF-κB or NF-κB mediated geneproducts. In a further embodiment, R₁, R₂, and R₃ are moieties such thatthe compound inhibits a cyclin kinase, particularly wherein the cyclinkinase is GSK-3β or CDK-1. In a further embodiment, R₁, R₂, and R₃ aremoieties such that the compound are inhibitors of checkpoint kinases,particularly wherein the kinase is CHK2. In a further embodiment, R₁,R₂, and R₃ are each selected from the group consisting of hydrogen,methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl, acyl,hydroxyl, amine, thiol, ester, ether, and amide. In a preferredembodiment, R₁, R₂, and R₃ are each hydrogen.

The present invention further provides a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are non-reactivegroups. In a preferred embodiment, R₁, R₂, and R₃ are each selected fromthe group consisting of hydrogen, methyl, alkyl containing 1 to 6 carbonatoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester, ether, andamide. In a particularly preferred embodiment, R₁, R₂, and R₃ are eachhydrogen.

The present invention further provides a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are non-reactivegroups. In a preferred embodiment, R₁, R₂, and R₃ are each selected fromthe group consisting of hydrogen, methyl, alkyl containing 1 to 6 carbonatoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester, ether, andamide. In a particularly preferred embodiment, R₁, R₂, and R₃ are eachhydrogen.

The present invention further provides a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are non-reactivegroups. In a preferred embodiment, R₁, R₂, and R₃ are each selected fromthe group consisting of hydrogen, methyl, alkyl containing 1 to 6 carbonatoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester, ether, andamide. In a particularly preferred embodiment, R₁, R₂, and R₃ are eachhydrogen.

The present invention further provides a process for the preparation ofa compound (IV) of the formula

herein R₁, R₂, and R₃ are which are non-reactive, which comprises (a)reacting a first compound (I) of the formula

with methyl sulfonic acid and phosphorus pentoxide at elevatedtemperatures to form a second compound (II) of the formula

(b) reacting compound (II) with phenyl oxazolone and a transition metalcatalyst in a solvent to form compound (III) of the formula

and (c) reacting compound (III) with thiourea and a base in a solvent toform the compound (IV). In a further embodiment of the process, in step(b) the transition metal catalyst is titanium chloride and the solventis tetrahydrofuran. In a further embodiment of the process, in step (c)the base is lithium hydride and the solvent is ethanol. In a furtherstill embodiment of the process, the compound (I) is formed by reactinga compound (V)

with an aqueous base solution, preferably wherein the base is lithiumhydroxide.

The present invention further provides a method for inhibiting a diseasein an animal, preferably a human, associated with a kinase or NF-κBactivation which comprises administering a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits the kinase, NF-κB activation or NF-κBmediated gene products, the compound being administered to the animal inan amount sufficient to substantially inhibit the disease. In a furtherembodiment, R₁, R₂, and R₃ are moieties such that the compound inhibitsa cyclin kinase, particularly wherein the cyclin kinase is GSK-3β orCDK-1. In a further embodiment, R₁, R₂, and R₃ are moieties such thatthe compounds are inhibitors of checkpoint kinases, particularly whereinthe kinase is CHK2. In a further embodiment, R₁, R₂, and R₃ are eachselected from the group consisting of hydrogen, methyl, alkyl containing1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester,ether, and amide. In a preferred embodiment, R₁, R₂, and R₃ are eachhydrogen.

In a preferred embodiment of the method, the disease associated withNF-κB activation is an inflammatory disorder. In a more preferredembodiment, the disease associated with NF-κB activation is selectedfrom the group consisting of rheumatoid arthritis, inflammatory boweldisease, asthma, dermatosis, autoimmune disease, tissue and organrejection, Alzheimer's disease, stroke, atherosclerosis, restenosis,cancer, viral infections, osteoarthritis, osteoporosis, and AtaxiaTelangiectasia.

In a preferred embodiment of the method, the disease associated withinhibition checkpoint kinases is cancer.

The present invention further provides a method for inhibitingproduction of cytokines in an animal, preferably a human, whichcomprises administering a compound of the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits the cytokines, the compound beingadministered to the animal in an amount sufficient to inhibit productionof the cytokines. In a further embodiment, R₁, R₂, and R₃ are moietiessuch that the compound inhibits a cyclin kinase, particularly whereinthe cyclin kinase is GSK-3β or CDK-1. In a further embodiment, R₁, R₂,and R₃ are moieties such that the compound are inhibitors of checkpointkinases, particularly wherein the kinase is CHK2. In a furtherembodiment, R₁, R₂, and R₃ are each selected from the group consistingof hydrogen, methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl,acyl, hydroxyl, amine, thiol, ester, ether, and amide. In a preferredembodiment, R₁, R₂, and R₃ are each hydrogen.

In a preferred embodiment of the method, the cytokines are produced byactivation of the NF-κB pathway. In a more preferred embodiment, thecytokines are selected from the group consisting of IL-2, IL-6, IL-8,TNF-α, and combinations thereof.

OBJECTS

It is an object of the present invention to provide small molecules andprocesses for synthesizing which are useful for treating inflammatorydiseases.

It is a further object of the present invention to provide smallmolecules and processes for synthesizing which are useful for treatingdiseases associated with NF-κB activation.

It is further an object of the present invention to provide smallmolecules and processes for synthesizing which are useful for treatingdiseases such as cancer.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of naturally-occurring hymenialdisine 1

FIG. 1B shows the structure for chemically-synthesized indoloazepinecompound 2.

FIG. 1C shows the structure for chemically-synthesized indoloazepinecompound 3.

FIG. 2A shows IL-2 production as measured by ELISA with PDTC.

FIG. 2B shows IL-2 production as measured by ELISA with hymenialdisine1.

FIG. 2C shows IL-2 production as measured by ELISA withchemically-synthesized indoloazepine compound 2.

FIG. 2D shows IL-2 production as measured by ELISA withchemically-synthesized indoloazepine compound 3.

FIG. 3 shows EMSA assays for NF-κB-DNA binding inhibition by compounds1-3. Lane 1: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract(+PMA/PHA)+Antibody p65; Lane 2: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (PMA/PHA); Lane 3: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (−PMA/−PHA); Lane 4: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (PMA/PHA)+5 μM PDTC; Lane 5: NF-κB consensusoligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+1 μM PDTC; Lane 6: NF-κBconsensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+5 μM compound 1;Lane 7: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+1μM compound 1; Lane 8: NF-κB consensus oligo (0.16 pmol/λ)+nuclearextract (PMA/PHA)+5 μM compound 2; Lane 9: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (PMA/PHA)+1 μM compound 2; Lane 10: NF-κBconsensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+5 μM compound 3;Lane 11: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA)+1μM compound 3.

FIG. 4 is a cartoon illustrating the role of NF-κB in activatingtranscription of particular genes such as those encoding variouscytokines.

FIG. 5 is a chart which compares the effect of compounds 1-3 on TNF-αproduction in THP-1 cells after stimulation with LPS.

FIG. 6A compares the cytotoxicity of hymenialdisine to compounds 2 and3. Number of cells were normalized on a scale of 100.

FIG. 6B shows the growth patterns in CEM cells at differentconcentrations of compound 2. Number of cells were normalized on a scaleof 100.

FIG. 7A is a graph showing inhibition of CDK1 and GSK-3β byhymenialdisine 1. x=CDK1 and ⋄=GSK-3β.

FIG. 7B is a graph showing inhibition of CDK1 and GSK-3β by compound 2.x=CDK1 and ⋄=GSK-3β.

FIG. 7C is a graph showing inhibition of CDK1 and GSK-3β by compound 3.x=CDK1 and ⋄=GSK-3β.

FIG. 8 is a table showing inhibition of the kinases: CK1(h), CK2(h),MEK1(h), PKCa(h), PKCbII(h), CHK1 and CHK2.

FIG. 9 is a graph showing inhibition of CHK1 and CHK2 by compound 2.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The present invention includes all hydrates, solvates, complexes, andprodrugs of the compounds of this invention. Prodrugs are any covalentlybonded compounds which in vivo releases the active parent drug havingthe formula The present invention further includes compounds andprodrugs of such compounds which have the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits NF-κB in human Jurkat Leukemia T-cellsactivated to transcribe NF-κB mediated gene transcription by phorbolmyristate acetate (PMA). In a further embodiment, R₁, R₂, and R₃ aremoieties such that the compound inhibits a cyclin kinase, particularlywherein the cyclin kinase is GSK-3β or CDK-1. In a further embodiment,R₁, R₂, and R₃ are each independently selected from the group consistingof methyl, alkyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cyclic, heterocyclic, substituted cyclic, andcombinations thereof; and, the R₁ and R₂ can be interconnected.Preferably, the R₁ and R₂ are interconnected members of an aryl orcyclic ring which can be substituted or comprise heteroatoms or both.Preferably, R₃ is hydrogen. The term “halo” includes F, Cl, Br, and I.The alkyl, aryl, acyl, cyclo includes substituted and heteroatom speciesas well as the unsubstituted species. If a chiral center or another formof an isomeric center is present in a compound of the present invention,all forms of such isomer or isomers, including enantiomers anddiastereomers, are intended to be covered herein. Compounds containing achiral center may be used as a racemic mixture, an enantiomericallyenriched mixture, or the racemic mixture may be separated usingwell-known techniques and an individual enantiomer may be used alone. Incases in which compounds have unsaturated carbon-carbon double bonds,both the cis (Z) and trans (E) isomers are within the scope of thisinvention. In cases wherein compounds may exist in tautomeric forms,such as keto-enol tautomers, each tautomeric form is contemplated asbeing included within this invention whether existing in equilibrium orpredominantly in one form.

The compounds disclosed herein are provided in organic solvents such asDMSO or other organic solvents which are pharmaceutically acceptable.The compounds disclosed herein are also provided as their amine acidsalts. For example, the compounds herein are prepared in an appropriatesolvent as taught herein. An excess of an acid such as acetic,hydrochloric, hydrobromic, hydrofluoric, maleic, methansulfonic,phosphoric, succinic, sulfuric, trifluoroacetic, or sulfuric acid isadded which produces the compound as its acid salt. The acid salts ofthe compounds are more soluble in water and other pharmaceuticallyacceptable aqueous solutions.

In light of the need for small molecule inhibitors of NF-κB mediatedgene transcription, the present invention provides chemical synthesisand methods of use for the compounds of the above formula. In apreferred embodiment, the compounds have the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits NF-κB in human Jurkat Leukemia T-cellsactivated to transcribe NF-κB mediated gene transcription by phorbolmyristate acetate (PMA). In a further embodiment, R₁, R₂, and R₃ aremoieties such that the compound inhibits a cyclin kinase, particularlywherein the cyclin kinase is GSK-3β or CDK-1. In a further embodiment,R₁, R₂, and R₃ are each independently selected from the group consistingof hydrogen, methyl, alkyl containing 1 to 6 carbon atoms, halo, aryl,acyl, hydroxyl, amine, thiol, ester, ether, and amide. In a preferredembodiment, R₁, R₂, and R₃ are each hydrogen.

In light of the need for small molecule inhibitors of checkpointkinases, the present invention provides chemical synthesis and methodsof use for the compounds of the above formula. In a preferredembodiment, the compounds have the formula

and acid amine salts thereof, wherein R₁, R₂, and R₃ are moieties suchthat the compound inhibits checkpoint kinases such as CHK1 and CHK2. Ina further embodiment, R₁, R₂, and R₃ are each independently selectedfrom the group consisting of hydrogen, methyl, alkyl containing 1 to 6carbon atoms, halo, aryl, acyl, hydroxyl, amine, thiol, ester, ether,and amide. In a preferred embodiment, R₁, R₂, and R₃ are each hydrogen.

In a preferred embodiment, the present invention provides theindoloazepine, compound 2, which has the formula

and acid amine salts thereof. A comparison of compound 2 tohymenialdisine 1 and compound 3, a methylated variant of compound 2, isshown in FIGS. 1A, 1B, and 1C.

Hymenialdisine 1 inhibits a wide variety of proinflammatory cytokinessuch as IL-2, IL-6, IL-8, and nitric oxides through inhibition of theNF-κB signaling pathway. Compound 2 also substantially inhibits NF-κBmediated cytokine production. The examples herein show that compound 2,in particular, inhibits IL-2 and TNF-α production with IC₅₀ valuessimilar to the IC₅₀ values for the naturally-occurring hymenialdisine 1.Compound 2 and its derivatives, including compounds with the aboveformula, substantially inhibits NF-κB mediated cytokine production,including IL-2, IL-6, IL-8, and nitric oxides. At the concentrationstested, compound 3 did not appear to have significant inhibitoryactivity. Because compounds 2 and 3, and derivatives thereof, includingcompounds with the above formula, can be chemically synthesized, theyprovide a convenient source of hymenialdisine-related small moleculeswhich can be used for treating a variety of inflammatory diseases whereit is desirable to inhibit NF-κB-mediated cytokine production. Thecompounds, like hymenialdisine, are also GSK-3β inhibitors (See WO03/027275 to Hellberg et al.).

The naturally-occurring hymenialdisine 1 and debromohymenialdisineinhibit the checkpoint kinases CHK1 and CHK2 at low micromolarconcentrations. Compound 2 is a much more potent and selective inhibitorof inhibited checkpoint kinases than the naturally-occurringhymenialdisine 1 and debromohymenialdisine. The examples herein showthat compound 2, in particular, inhibits CHK1 with an IC₅₀ value of 237nanoMolar concentration and inhibits CHK2 with an IC₅₀ value of 8nanoMolar concentration. This shows that the synthetically preparedindoloazepines have an improved overall kinase profile and significantlyimproves its kinase selectivity.

Therefore, the present invention provides pharmaceutical compositionscomprising one or more of the compounds with the above formula which areinhibitors of transcription factor NF-κB, and methods for treatingdiseases in which activation of NF-κB or activity of GSK-3β isimplicated. More specifically, the present invention providescompositions and methods of treatment of a variety of diseasesassociated with NF-κB or GSK-3β activation or GSK-3β inflammatorydisorders; particularly rheumatoid arthritis, inflammatory boweldisease, and asthma; dermatosis, including psoriasis and atopicdermatitis; autoimmune diseases; tissue and organ rejection; Alzheimer'sdisease; stroke; atherosclerosis; restenosis; cancer, includingHodgkin's disease; a variety of viral infections, including AIDS;osteoarthritis; osteoporosis; glaucoma; and, Ataxia Telangiectasia byadministering to an animal or human in need thereof a compoundcomprising the above formula.

Therefore, the present invention also provides pharmaceuticalcompositions comprising one or more of the compounds with the aboveformula which are inhibitors of checkpoint kinases, and methods fortreating diseases in which checkpoint kinases are implicated. Morespecifically, the present invention provides compositions and methods oftreatment of a variety of diseases associated with checkpoint kinasessuch as cancer.

Without intending to be bound by any particular theory, the inhibitionof NF-κB by the compounds of the above formula is believed to be exertedin the nucleus of the cell as shown in FIG. 4. Under normal cellularconditions, NF-κB is sequestered in a complex with IκB. In response to asignal by an unknown pathway, a kinase phosphorylates IκB in thecytoplasm. IκB then degrades which releases the NF-κB. The NF-κB entersthe nucleus and is believed to undergo additional phosphorylation byGSK-3β. The phosphorylated NF-κB can then bind to the cellular DNA whichstimulates transcription of genes encoding cytokines such as IL-1, IL-2,IL-6, IL-8, and TNF-α. It also has a role in stimulating transcriptionof genes which are involved in cell growth such as DNA repair, celldivision, and cell survival. The compounds of the above formula likelyinhibit the phosphorylation of NF-κB by GSK-3 by binding to the ATPbinding pocket of the GSK-3β. As a result, cytokine production isinhibited and cell growth is inhibited. The above scheme is to becontrasted with the activity of other compounds such as imidizolineswhich inhibit NF-κB in the cytoplasm, not in the nucleus.

The synthesis of the indol-aldisine or indoloazepine skeleton (compounds2 and 3 shown in FIG. 1) is shown in Scheme 1 and described inExample 1. It was achieved via a modified route as reported in thesynthesis of aldisine (Annoura and Tatsuoka, Tet. Let. 36: 413-416(1995); Mizuno et al., Chem. Pharm. Bull. 47: 246-256 (1999); Cho etal., J. Heterocycl. Chem. 34: 87-91 (1997); Xu et al., J. Org. Chem. 62:456-464 (1997)).

Starting with the commercially available 2-indolecarboxylic acid,condensation with the ethyl ester of β-alanine in the presence of EDCIand DMAP, provided the indole 4. Methylation of the indole nitrogen withMeI and K₂CO₃ proceeded in near quantitative yields, rendering theN-methyl indole, which was used for the synthesis of 3. Hydrolysis ofesters 4 and 5 followed by the P₂O₅/MeSO₃H mediated cyclization providedthe key intermediate aldisine derivatives 8 and 9.

The direct condensation of the aldisine derivatives 8 or 9 with theimidazolone precursor (Scheme 2) proved to be unsuccessful similar toearlier reports with the pyrrolazepines (Scheme 2) (Prager and Tsopelas,Aust. J. Chem. 43: 367-374 (1990); Prager and Tsopelas, Aust. J. Chem.45: 1771-1777 (1992)). However, TiCl₄ mediated Aldol condensation withthe phenyl oxazolone 12 provided the oxazolone successfully in 55% and56% yield for compounds 10 and 11, respectively. Treatment of theoxazolone derivatives 10 and 11 with the thiourea under basic conditionsprovided the final products, indoloazepines (compounds) 2 and 3 inmodest yields.

Hymenialdisine 1 and compounds 2 and 3 were evaluated for theiranti-inflammatory activity by examining the transcriptional activity ofNF-κB in human Jurkat leukemia T-cells and THP-1 cells (human monocyticleukemia cell line) (Examples 2-5). Exposure of human Jurkat leukemiaT-cells to phorbol myristate acetate (PMA/PHA) and THP-1 cells tolipopolysaccharide (LPS), activates NF-κB mediated gene transcription ofseveral pro-inflammatory cytokines, including IL-2, IL-6, IL-8 and TNF-α(Baldwin, Ann. Rev. Immunol. 14: 649-683 (1996); Grilli et al., Int.Rev. Cytol. 143: 1-62 (1993)). The anti-oxidant and non-selective NF-κBinhibitor, pyrrolidinedithiocarbamate (PDTC), has been reported torepress activation of NF-κB and was used as a control in all experimentsin addition to an authentic sample of the natural product,hymenialdisine (Grilli et al., Int. Rev. Cytol. 143: 1-62 (1993); Epinatand Gilmore, Oncogene 18: 6896-6909 (1999); Cuzzocrea et al., Br. J.Pharmacol. 135: 496-510 (2002)). The effect of hymenialdisine andcompounds 2 and 3 on NF-κB's transcriptional activity was evaluated bymeasuring the level of IL-2 and TNF-α production using a competitiveenzyme immunoassay (EIA). The inhibition of IL-2 was evaluated in Jurkatcells after PMA/PHA activation (Mahon and O'Neill, J. Biol. Chem. 270:28557-28564 (1995); Lindgren et al., Molec. Immunol. 38V 267-277(2001)). The inhibition of TNF-α was evaluated in THP-1 cells after LPSactivation (Aikawa et al., Inflamm. Res. 51: 188-194 (2002)). NF-κBmediated transcription of IL-2 production was examined by exposing thecells to PDTC or compounds 1-3, 30 minutes prior to PMA activation.After 24 hours, the cell free supernatant fractions were collected andsubjected to EIA for the quantification of total IL-2 production.

Compound 2 and the natural product hymenialdisine exhibited potentinhibition of IL-2 and TNF-α production, whereas the N-methyl compound 3had substantially less IL-2 or TNF-α inhibitory activity. Hymenialdisineand compound 2 were found to inhibit DNA binding by NF-κB. Gelelectrophoresis assays indicated that none of the compounds had anynotable effect on the DNA binding of the transcription factor AP-1.

Hymenialdisine and compounds 2 and 3 were tested for inhibition of thecyclin dependent kinases CDK1 and GSK-3β using the method of Meijer etal., Chem. Biol. 7: 51-63 (2000). Like hymenialdisine, compound 2inhibited CDK1 (IC₅₀=0.4 μM) and GSK-3β (IC₅₀=150 ηM). The inhibitoryactivity of compound 3 was less pronounced.

Thus, compounds which have the formula

wherein R₁, R₂, and R₃ are moieties such that the compound inhibitsNF-κB in human Jurkat Leukemia T-cells activated to transcribe NF-κBmediated gene transcription by phorbol myristate acetate (PMA) provide anew synthetically readily available scaffold for further optimizationfor cytokine inhibition in animals or humans in need thereof. In afurther embodiment, R₁, R₂, and R₃ are moieties such that the compoundinhibits a cyclin kinase, particularly wherein the cyclin kinase isGSK-3β or CDK-1. In a further embodiment, R₁, R₂, and R₃ are eachindependently selected from the group consisting of hydrogen, methyl,alkyl containing 1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine,thiol, ester, ether, and amide. It is further embodiment, it ispreferable that R₃ is hydrogen. In a particularly preferred embodiment,the compound is compound 2.

Hymenialdisine and compound 2 were tested for inhibition of thecheckpoint kinases CHK1 and CHK2 by UpState, Inc. Compound 2 inhibitedCHK1 (IC₅₀=237 nM) and CHK2 (IC₅₀=8 nM) and was many folds more potentthan the naturally-occurring hymenialdisines.

Thus, compounds which have the formula

wherein R₁, R₂, and R₃ are moieties such that the compound inhibitscheckpoint kinases provide a new synthetically readily availablescaffold for further optimization for anticancer therapy in animals orhumans in need thereof. In a further embodiment, R₁, R₂, and R₃ are eachindependently selected from the group consisting of hydrogen, methyl,alkyl containing 1 to 6 carbon atoms, halo, aryl, acyl, hydroxyl, amine,thiol, ester, ether, and amide. In a further embodiment, it ispreferable that R₃ is hydrogen. In a particularly preferred embodiment,the compound is compound 2.

The present invention further provides a pharmaceutical compositionwhich comprises one or more compounds according to the above formulaeand a pharmaceutically acceptable carrier, diluent, or excipient. Thus,the compound may be used in the manufacture of a medicament.Pharmaceutical compositions of the compound synthesizes as describedherein can be formulated as solutions or lyophilized powders forparenteral administration. The powders can be reconstituted by additionof a suitable diluent or other pharmaceutically acceptable carrier priorto use. The liquid formulation can be a buffered, isotonic, aqueoussolution. Examples of suitable diluents are normal isotonic salinesolution, standard 5% dextrose in water, or buffered sodium or ammoniumacetate solution. Such formulations are especially suitable forparenteral administration, but may also be used for oral administrationor contained in a metered dose inhaler or nebulizer for insulation. Inparticular embodiments, it can be desirable to further add one or moreexcipients selected from the group consisting of polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,sodium chloride, and sodium citrate.

Alternately, one or more of the above compounds may be encapsulated,tableted or prepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers can be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Solid carriers include starch, lactose, calcium sulfatedihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin,acacia, agar, or gelatin. Liquid carriers include syrup, peanut oil,olive oil, saline, and water. The carrier can also include a sustainedrelease material such as glyceryl monostearate or glyceryl distearate,alone or with a wax. The pharmaceutical preparations are made followingthe conventional techniques of pharmacy such as milling, mixing,granulating, and compressing, when necessary, for tablet forms; ormilling, mixing, and filling for hard gelatin capsule forms. When aliquid carrier is used, the preparation will be in the form of a syrup,elixir, emulsion, or an aqueous or non-aqueous suspension. Such a liquidformulation can be administered directly or filled into a soft gelatincapsule.

For rectal administration, one or more of the above compounds can alsobe combined with excipients such as cocoa butter, glycerin, gelatin, orpolyethylene glycols and molded into a suppository.

The methods of the present invention further include topicaladministration of one or more of the above compounds. By topicaladministration is meant non-systemic administration, including theapplication of a compound of the invention externally to the epidermis,to the buccal cavity, and instillation into the ear, eye, and nose,wherein the compound does not significantly enter the blood stream. Bysystemic administration is meant oral, intravenous, intraperitoneal, andintramuscular administration. The amount of a compound of the inventionrequired for therapeutic or prophylactic effect upon topicaladministration will, of course, vary with the compound chosen, thenature and severity of the condition being treated and the animal orhuman undergoing treatment, and is ultimately at the discretion of thephysician.

The topical formulations of the present invention, both for veterinaryand for human medical use, comprise one or more of the above compoundstogether with one or more acceptable carriers therefor, and optionallyany other therapeutic ingredients.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required such as: liniments, lotions,creams, ointments, or pastes, and drops suitable for administration tothe eye, ear, or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving one ormore of the above compounds in a suitable aqueous solution of abactericidal and/or fungicidal agent and/or any other suitablepreservative, and preferably including a surface active agent. Theresulting solution can then be clarified by filtration, transferred to asuitable container which is then sealed and sterilized by autoclaving ormaintaining at 90-100° C. for half an hour. Alternatively, the solutioncan be sterilized by filtration and transferred to the container by anaseptic technique. Examples of bactericidal and fungicidal agentssuitable for inclusion in the drops are phenylmercuric nitrate oracetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidineacetate (0.01%). Suitable solvents for the preparation of an oilysolution include glycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion can comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments, or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the compound in finely-dividedor powdered form, alone or in solution or suspension in an aqueous ornon-aqueous fluid, with the aid of suitable machinery, with a greasy ornon-greasy basis. The basis may comprise hydrocarbons such as hard, softor liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; anoil of natural origin such as almond, corn, arachis, castor or oliveoil; wool fat or its derivatives, or a fatty acid such as stearic oroleic acid together with an alcohol such as propylene glycol ormacrogols. The formulation may incorporate any suitable surface activeagent such as an anionic, cationic or non-ionic surface active agentsuch as sorbitan esters or polyoxyethylene derivatives thereof.Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredientssuch as lanolin, may also be included.

The present invention also provides methods of treatment of diseasesassociated with NF-κB activation, which methods comprise administeringto an animal, particularly an animal, most particularly a human in needthereof, one or more of the above compounds. The present inventionparticularly provides methods for treating inflammatory disorders;particularly rheumatoid arthritis, inflammatory bowel disease, andasthma; dermatosis, including psoriasis and atopic dennatitis;autoimmune diseases; tissue and organ rejection; Alzheimer's disease;stroke; atherosclerosis; restenosis; cancer, including Hodgkins disease;and certain viral infections, including AIDS; osteoarthritis;osteoporosis; and Ataxia Telangiectasia.

For acute therapy, parenteral administration of one or more of the abovecompounds is preferred. An intravenous infusion of the compound can bein 5% dextrose in water or normal saline, or a similar formulation withsuitable excipients. Typically, the parenteral dose will be at aconcentration effective to inhibit activation of NF-κB. The preciseamount of an inventive compound which is therapeutically effective, andthe route by which such compound is best administered, is readilydetermined by one of ordinary skill in the art by comparing the bloodlevel of the agent to the concentration required to have a therapeuticeffect.

The compounds of the above formula can also be administered orally tothe animal or human, in a manner such that the concentration of drug issufficient to inactivate NF-κB or to achieve any other therapeuticindication as disclosed herein. Typically, a pharmaceutical compositioncontaining one or more of the above compounds is administered in amountwhich for the body weight of the animal or human inactivates the NF-κB.

One or more of the above compounds can also be administered topically tothe animal or human, in a manner such that the concentration of drug issufficient to inhibit NF-κB.

In some embodiments of the invention, the compound of the above formulais used in combination with one or more other anti-inflammatory,anti-viral, anti-fungal, amoebicidal, trichomonocidal, analgesic,anti-neoplastic, anti-hypertensives, anti-microbial and/or steroid drugsor potentiators. Such drugs include triprolidine or its cis-isomer whichis used in combination with chemotherapeutic agents; a compound of theabove formula and procodazole, 1H-benzimidazole-2-propanoic acid;[β-(2-benzimidazole)propionic acid 2-(2-carboxyethyl)benzimidazole;propazol] which is a non-specific immunoprotective agent active againstviral and bacterial infections that is used with the compound of theabove formula; or a compound of the above formula and aplatinum-containing drug such as cisplatin which binds DNA whichinterferes with its DNA repair mechanism and thereby causing cellulardeath. Other drugs which can be used with a compound of the aboveformula, and optionally another chemotherapeutic agent, in the methodsof the invention include macrophage colony-stimulating factor (M-CSF),7-thia-8-oxoguanosine, 6-mercaptopurine, vitamin A (retinol), and otherknown anti-tumor potentiators which can be used in conjunction with thecompounds of the above formula include, monensin, an anti-senseinhibitor of the RAD51 gene, bromodeoxyuridine, dipyridamole,indomethacin, a monoclonal antibody, an anti-transferrin receptorimmunotoxin, metoclopramide,N-solanesyl-N,N′-bis(3,4-dimethoxybenzyl)ethylenediamine, leucovorin,heparin, N-[4-[(4-fluorphenyl)sulfonly]phenyl]acetamide, heparinsulfate, cimetidine, a radiosensitizer, a chemosensitizer, a hypoxiccell cytotoxic agent, muramyl dipeptide, vitamin A, 2′-deoxycoformycin,a bis-diketopiperazine derivative, and dimethyl sulfoxide otheranti-tumor potentiators.

The chemotherapeutic agents which can be used with a compound of theabove formula and an optional potentiator are generally grouped asDNA-interactive agents, antimetabolites, tubulin-interactive agents,hormonal agents, and others such as asparaginase or hydroxyarea. Each ofthe groups of chemotherapeutic agents can be further divided by type ofactivity or compound.

DNA-interactive agents include the alkylating agents, for example,cisplatin, cyclophosphamide, altretamine; the DNA strand-breakingagents, such as bleomycin; the intercalating topoisomerase IIinhibitors, for example, dactinomycin and doxorubicin; thenonintercalating topoisomerase II inhibitors, such as etoposide andteniposide; and the DNA minor groove binder plicamycin.

The alkylating agents form covalent chemical adducts with cellular DNA,RNA, and protein molecules and with smaller amino acids, glutathione andsimilar chemicals. Generally, these alkylating agents react with anucleophilic atom in a cellular constituent, such as an amino, carboxyl,phosphate, sulfhydryl group in nucleic acids, proteins, amino acids, orglutathione. The mechanism and the role of these alkylating agents incancer therapy is not well understood. Typical alkylating agentsinclude: nitrogen mustards, such as chlorambucil, cyclophosphamide,isofamide, mechlorethamine, melphalan, uracil mustard; aziridine such asthiotepa; methanesulphonate esters such as busulfan; nitroso ureas, suchas carmustine, lomustine, streptozocin; platinum complexes, such ascisplatin, carboplatin; bioreductive alkylator, such as mitomycin, andprocarbazine, dacarbazine, and altretamine.

DNA strand breaking agents include Bleomycin. DNA topoisomerase IIinhibitors include the following: intercalators, such as amsacrine,dactinomycin, daunorubicin, doxorubicin, idarubicin, and mitoxantrone;and nonintercalators, such as etoposide and teniposide. The DNA minorgroove binder is Plicamycin.

The antimetabolites interfere with the production of nucleic acids byone or the other of two major mechanisms. Some of the drugs inhibitproduction of the deoxyribonucleoside triphosphates that are theimmediate precursors for DNA synthesis, thus inhibiting DNA replication.Some of the compounds are sufficiently like purines or pyrimidines to beable to substitute for them in the anabolic nucleotide pathways. Theseanalogs can then be substituted into the DNA and RNA instead of theirnormal counterparts. The antimetabolites useful herein include: folateantagonists such as methotrexate and trimetrexate; pyrimidineantagonists, such as fluorouracil, fluorodeoxyunridine, CB3717,azacitidine and floxuridine; purine antagonists such as mercaptopurine,6-thioguanine, pentostatin; sugar modified analogs such as cytarabineand fludarabine; and ribonucleotide reductase inhibitors such ashydroxyurea.

Tubulin interactive agents act by binding to specific sites on tubulin,a protein that polymerizes to form cellular microtubules. Microtubulesare critical cell structure units. When the interactive agents bind onthe protein, the cell can not form microtubules tubulin interactiveagents include colchicine, vincristine and vinblastine, both alkaloidsand paclitaxel and cytoxan.

Hormonal agents are also useful in the treatment of cancers and tumors.They are used in hormonally susceptible tumors and are usually derivedfrom natural sources. These include estrogens, conjugated estrogens andethinyl estradiol and diethylstilbesterol; chlortrianisen andidenestrol; progestins such as hydroxyprogesterone caproate.medroxyprogesterone, and megestrol; and androgens such as testosterone,testosterone propionate, fluoxymesterone, and methyltestosterone.

Adrenal corticosteroids are derived from natural adrenal cortisol orhydrocortisone. They are used because of their anti inflammatorybenefits as well as the ability of some to inhibit mitotic divisions andto halt DNA synthesis. These compounds include, prednisone,dexamethasone, methylprednisolone, and prednisolone.

Leutinizing hormone releasing hormone agents or gonadotropin-releasinghormone antagonists are used primarily the treatment of prostate cancer.These include leuprolide acetate and goserelin acetate. They prevent thebiosynthesis of steroids in the testes.

Antihormonal antigens include: antiestrogenic agents such as tamoxifen;antiandrogen agents such as flutamide; and antiadrenal agents such asmitotane and aminoglutethimide.

Hydroxyurea, which appears to act primarily through inhibition of theenzyme ribonucleotide reductase, can also be used in combination withthe compound of the above formula.

Asparaginase is an enzyme which converts asparagine to nonfunctionalaspartic acid and thus blocks protein synthesis in the tumor.Asparaginase can also be used in combination with the compound of theabove formula to treat cancer.

Other chemotherapeutic benzimidazoles and griseofulvin can also be usedin combination with the compound of the above formula and optionally apotentiator to treat or inhibit the growth of cancer or extend the lifespan of a animal or human having cancer.

The amount and identity of a chemotherapeutic agent that is used with acompound of the above formula in the methods of the invention will varyaccording to cellular response, patient response and physiology, typeand severity of side effects, the disease being treated, the preferreddosing regimen, patient prognosis, or other such factors.

The compound of the above formula can be used in combination with one ormore other agents or combination of agents known to possessanti-leukemia activity including, by way of example, α-interferon;interleukin-2; cytarabine and mitoxantrone; cytarabine and daunorubicinand 6-thioguanine; cyclophosphamide and 2-chloro-2′-deoxyadenosine;VP-16 and cytarabine and idorubicin or mitoxantrone; fludarabine andcytarabine and γ-CSF; chlorambucil; cyclophosphamide and vincristine and(prednisolone or prednisone) and optionally doxorubicin; tyrosine kinaseinhibitor; an antibody; glutamine; clofibric acid; all-trans retinoicacid; ginseng diyne analog; KRN8602 (anthracycline drug); temozolomideand poly(ADP-ribose) polymerase inhibitors; lysofylline; cytosinearabinoside; chlythorax and elemental enteral diet enriched withmedium-chain triglycerides; amifostine; gilvusmycin; or a hot waterextract of the bark of Acer nikoense.

The compounds of the above formula can further be administered to ananimal or human with one or more imidazolines and optionally one or moreof the above drugs or potentiators as a treatment for cellularproliferative diseases. As used herein, antiproliferative agents arecompounds, which induce cytostasis or cytotoxicity. Cytostasis is theinhibition of cells from growing while cytotoxicity is defined as thekilling of cells. Specific examples of antiproliferative agents includeantimetabolites, such as methotrexate, 5-fluorouracil, gemcitabine,cytarabine; anti-tubulin protein agents such as the vinca alkaloids,paclitaxel, colchicine; hormone antagonists, such as tamoxifen, LHRHanalogs; and nucleic acid damaging agents such as the alkylating agentsmelphalan, BCNU, CCNU, thiotepa, intercalating agents such asdoxorubicin and metal coordination complexes such as cisplatin andcarboplatin.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

This example shows the synthesis of compounds 2 and 3 as shown in FIGS.1B and 1C and Scheme 1.

Reactions were carried out in oven-dried glassware under nitrogenatmosphere, unless otherwise noted. All commercial reagents were usedwithout further purification. All solvents were reagent grade. THF wasfreshly distilled from sodium/benzophenone under nitrogen. CH₂Cl₂ wasfreshly distilled from CaH₂ under nitrogen. All reactions weremagnetically stirred and monitored by thin layer chromatography withAnaltech 0.25-mm pre-coated silica gel plates (Analtech, Inc., Newark,Del.). Column chromatography was carried out on silica gel 60 (230-400mesh) supplied by EM Science (END Chemicals, Inc., Gibbstown, N.J.).Yields refer to chromatographically and spectroscopically pure compoundsunless otherwise stated. Infrared spectra were recorded on a NicoletIR/42 spectrometer. Proton and carbon NMR spectra were recorded on aVarian Gemini-300 spectrometer or a Varian VXR-500 spectrometer (VarianMedical Systems, Inc., Palo Alto, Calif.). Chemical shifts were reportedrelative to the residue peaks of solvent chloroform (δ 7.24 for ¹H and δ77.0 for ¹³C) and dimethyl sulfoxide (δ 2.49 for ¹H and δ 39.5 for ¹³C).High-resolution mass spectra were obtained at the Mass SpectrometryLaboratory of the University of South Carolina, Department of Chemistry& Biochemistry with a Micromass VG-70S mass spectrometer. Gaschromatography/low-resolution mass spectra were recorded on a HewlettPackard 5890 Series II gas chromatograph connected to a TRIO-1 EI massspectrometer. All regents were obtained from Aldrich Chemical Co., St.Louis, Mo., and used as received.

Synthesis of 3-[(1H-indole-2-carbonyl)-amino]-propionic acid ethylester, compound 4, was as follows. To a mixture of 2-indolecarboxylicacid (161 mg, 1.0 mmol), DMAP (200 mg, 6 mmol) and alanine ethyl esterhydrochloride (168 mg, 1.1 mmol) in 10 mL anhydrous CH₂Cl₂ was addedEDCI (215 mg, 1.1 mmol) at 0° C. The mixture then was stirred at 0° C.for 4 hours and at room temperature for 20 hours. The mixture was washedwith water (20 mL) and 10% HCl (10 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated to yield product 4 as a white solid (230 mg,88.5%). Mp 158-160° C. ¹H NMR: (300 MHz, CDCl₃) δ 1.28 (t, 3H, J=7.2Hz), 2.69 (t, 2H, J=5.7 Hz), 3.78 (q, 2H, J=6.0 Hz), 4.18 (q, 2H, J=6.9,7.2 Hz), 6.89 (s, 1H), 7.05 (s, 1H), 7.13-7.68 (m, 4H), 9.86 (s, 1H).¹³C NMR: (74.47 MHz, CDCl₃) δ 14.1, 33.9, 34.9, 60.8, 102.2, 111.9,120.4, 121.8, 124.3, 127.5, 130.5, 136.4, 161.7, 172.8. IR: (NaCl) 3377,3352, 1716, 1624, 1552, 1325, 1207, 748 cm⁻¹. HRMS m/e calcd forC₁₄H₁₆N₂O₃ (M) 260.1161, found 260.1159.

Synthesis of 3-[(1-methyl-1H-indole-2-carbonyl)-amino]-propionic acidethyl ester, compound 5, was as follows. A solution of compound 4 (0.5g, 1.9 mmol), K₂CO₃ (1.06 g, 7.68 mmol) and iodomethane (0.29 mL, 4.64mmol) in acetonitrile (15 mL) was stirred at reflux for 24 hours. Themixture was cooled, filtered and the filtrate was evaporated in vacuo.The crude material was further purified by column chromatography onsilica gel (CH₂Cl₂—AcOEt 3:1) to yield compound 5 as white solid (512mg, 98%); Mp 75-77° C. ¹H NMR: (300 MHz, CDCl₃) δ 1.28 (t, 3H, J=7.2Hz), 2.67 (m, 2H), 3.72 (m, 2H), 4.07 (s, 3H), 4.22 (q, 2H, J=6.9 Hz),6.86 (s, 1H), 7.05-7.65 (m, 4H). ¹³C NMR: (74.47 MHz, CDCl₃) δ 14.1,31.4, 33.9, 34.8, 60.7, 103.8, 110.1, 120.3, 121.7, 123.9, 125.9, 131.8,138.9, 162.4, 172.6. IR (NaCl): 3263, 1732, 1630, 1552, 1462, 1392,1321, 1182, 746 cm⁻¹. HRMS m/e calcd for C₁₅H₁₈N₂O₃ (M) 274.1317, found274.1315.

Synthesis of 3-[(1-methyl-1H-indole-2-carbonyl)-amino]-propionic acid,compound 7, was as follows. A solution of compound 5 (420 mg, 1.53 mmol)and LiOH (129 mg, 3.07 mmol ) in ethanol (14 mL) was stirred at roomtemperature for 18 hours. After cooling, the solvent was evaporated invacuo. The residue was dissolved in water and acidified with 1N HCL topH=3 to afford white precipitate, which isolated by filtration andwashed with water to provide compound 7 (360 mg, 95%); Mp 160-162° C. ¹HNMR: (300 MHz, d⁶-DMSO) δ 2.49 (t, 2H, J=7.2 Hz), 3.46 (q, 2H, J=6.6Hz), 3.97 (s, 3H), 7.07-7.64 (m, 5H), 8.54 (t, 1H, J=5.7 Hz). ¹³C NMR:(74.47 MHz, d⁶-DMSO) δ 31.3, 33.8, 35.2, 104.3, 110.5, 120.1, 121.5,123.5, 125.6, 132.1, 138.4, 161.9, 173.0. IR (NaCl): 3385, 2966, 1713,1603, 1550, 1468, 1425, 1400, 1282, 1228, 1192, 910, 748 cm⁻¹. HRMS m/ecalcd for C₁₃H₁₄N₂O₃ (M) 246.1004, found 246.1016.

Synthesis of10-methyl-3,4-dihydro-2H,10H-azepino[3,4-b]indole-1,5-dione, compound 9,was as follows. Compound 7 (2.16 g, 8.78 mmol) was added to a clearsolution of P₂O₅ (3.49 g, 12.3 mmol) in MeSO₃H (21 mL) at 60° C. Themixture was heated to 110° C. for 1 hour, after which the mixture wascooled to room temperature. The reaction mixture was poured intoice-water, stirred for 30 minutes, filtered and washed with water, toprovide compound 9 (1.143 g) as a solid (57%). Mp 200-205° C. ¹H NMR:(500 MHz, d⁶-DMSO) δ 2.77 (t, 2H, J=5.5 Hz), 3.39 (t, 2H, J=5.5, 4.5Hz), 3.99 (s, 3H), 7.27 (t, 1H, J=7.0, 8.0 Hz), 7.37 (t, 1H, J=7.5 Hz),7.62 (d, 1H, J=8.0 Hz), 8.27 (d, 1H, J=8.0 Hz), 8.77 (t, 1H, J=5.0, 6.0Hz); ¹³C NMR: (124.1 MHz, d⁶-DMSO) δ 33.0, 37.3, 45.6, 111.6, 115.5,123.4, 123.8, 125.3, 125.7, 135.4, 138.7, 163.1, 196.4. IR (NaCl): 3204,3000, 2924, 1662, 1641, 1506, 1473, 1371, 724 cm⁻¹; HRMS m/e calcd forC₁₃H₁₂N₂O₂ (M) 228.0899, found 228.0887.

Synthesis of10-methyl-5-(5-oxo-2-phenyl-oxazol-4-ylidene)-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 11 was as follows. A solution of TiCl₄ (1.54 mL, 14 mmol) inCH₂Cl₂ (10 mL) was added into THF (10 mL) at 0° C. Compound 9 (802 mg,3.5 mmol) and 2-phenylazlactone (1.13 g, 7 mmol) were added to thismixture. The mixture was stirred at 0° C. for 20 minutes after whichpyridine (1.13 mL, 14 mmol) was added. The reaction mixture was stirredat 0° C. for an additional 2 hours, after which it was allowed to stirovernight at room temperature. NH₄Cl (80 mL, sat. solution in water) wasadded and the mixture was stirred for 10 minutes. The mixture wassubsequently extracted with ethyl acetate (3 times), the extractscombined, dried with anhydrous Na₂SO₄, filtered, concentrated and theresidue was purified with column chromatography on silica gel (ethylacetate-hexane 8:2) to yield compound 11 (0.73 g, 56%) as yellow solid.Mp: 189-192° C. ¹H NMR: (300 MHz, CDCl₃) δ 3.54 (t, 2H, J=3.3, 3.6 Hz),3.59 (t, 2H, J=2.7, 3.3 Hz), 4.09 (s, 3H), 6.94 (s, 1H), 7.24-7.27 (m,1H), 7.38-7.49 (m, 5H), 7.78 (d, 1H, J=4.8 Hz), 7.92 (d, 2H, J=6.0 Hz);¹³C NMR: (74.47 MHz, CDCl₃) δ 31.9, 38.2, 38.4, 110.1, 115.8, 121.3,124.3, 125.0, 125.1, 125.8, 127.5, 128.7, 130.0, 131.7, 132.4, 138.5,144.5, 159.4, 165.3, 165.9. IR: (NaCl) 1784, 1753, 1662, 1633, 1473cm⁻¹; LRMS (EI): 371.1(M); HRMS m/e calcd for C₂₂H₁₇N₃O₃ (M) 371.1270,found 371.1268.

Synthesis of5-(2-amino-5-oxo-1,5-dihydro-imidazol-4-ylidene)-10-methyl-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 3, was as follows. LiH (48 mg, 6 mmol) was dissolved in ethanol(150 mL) and to this solution was added compound 11 (371 mg, 1.0 mmol)and S-benzylisothiouronium chloride (1.01 g, 5 mmol). The reaction wasrefluxed 48 hours, after which the solvent was evaporated in vacuo. 50mL ethanol was added to the reaction and was evaporated. This wasrepeated 3 times. Ethanol (15mL) was added again and the reaction wasrefluxed for 3 hours, after which the solvent was distilled off invacuum. 1N HCL_((aq)) (50 mL) was added and the product was extractedwith n-butanol (3×50 mL). The extracts were combined and washed withbrine (3×20 mL), dried, concentrated and the product was purified bycolumn chromatography on silica gel (CH₂Cl₂: MeOH:NH₃.H₂O, 3:1:0.1) toyield compound 3 as light yellow solid (96 mg, 31%) Mp: >260° C. ¹H NMR:(300 MHz, d⁶-DMSO) δ 2.99-3.48 (m, 4H) 3.92 (s, 3H), 7.19 (t, 1H, J=7.2Hz), 7.36 (t, 1H, J=6.6 Hz), 7.55 (d, 1H, J=8.1 Hz), 7.65 (d, 1H, J=8.4Hz), 8.57 (t, 1H, J=5.1 Hz), 9.15-9.25 (br-s,1H), 10.48 (s, 1H); ¹³CNMR: (74.47 MHz, d⁶-DMSO) δ 32.1, 37.1, 38.4, 111.7, 112.6, 122.1,122.2, 123.5, 123.7, 125.1, 128.1, 132.8, 138.5, 154.6, 163.0, 165.1;IR: (KBr-pellet) 3211, 1699, 1635, 1508, 1477 cm⁻¹; LRMS (EI): 308.7(M); HRMS m/e calcd for C₁₆H₁₅N₅O₂ (M) 309.1304, found 309.1291.

Synthesis of 3-[(1H-indole-2-carbonyl)-amino]-propionic acid ethylester, compound 6, was as follows. A mixture of compound 4 (1.838 g,7.38 mmol ) and LiOH (0.6 g, 14.1 mmol) was stirred at room temperaturein ethanol (50 mL) for 20 hours, the ethanol was evaporated to drynessin vacuo. The residue was dissolved in water (50 mL), acidified thesolution to pH=1 with HCL (aq), at which a white solid precipitated. Themixture was allowed to stand at 0° C. for 30 minutes, after which theproduct was filtered of, dried in vacuum, to yield compound 6 (1.45 g,84%). Mp 232° C. ¹H NMR: (300 MHz, d⁶-DMSO) δ 2.52 (t. 2H, J=6.9 Hz),3.47 (q, 2H, J=6.6, 6.0 Hz), 6.98 (t, 1H, J=7.2 Hz), 7.10 (s, 1H), 7.13(t, 1H, J=7.2 Hz), 7.41 (1H, J=8.1 Hz), 7.57 (1H, J=8.1 Hz), 8.52 (s,1H), 11.55 (s, 1H), 12.25 (s, 1H); ¹³C NMR: (74.47 MHz) (d⁶-DMSO) δ34.6, 35.9, 103.2, 112.9, 120.3, 122.1, 123.9, 127.8, 132.4, 137.1,161.8, 173.4; IR (NaCl) : 3422, 3273, 1745, 1707, 1643, 1549, 1417,1341, 1259, 746 cm⁻¹; HRMS m/e calcd for C₁₂H₁₂N₂O₃ (M) 232.0848, found232.0844.

Synthesis of 3,4-dihydro-2H,10H-azepino[3,4-b]indole-1,5-dione, compound8, was as follows. Compound 6 (116 mg, 0.5 mmol) was added to a clearsolution of P₂O₅ (232 mg, 0.8 mmol ) in MeSO₃H (1.57 mL) at 60° C. Themixture was heated to 110° C. for 1.5 hour, after which the mixture wascooled to room temperature. The reaction mixture was poured intoice-water, stirred for 30 minutes, filtered, dissolved in acetone (100mL), filtered again, and the filtrate was concentrated. The product wasfurther purified by column chromatography on silica gel to yieldcompound 8 (88 mg, 82%) Mp: 257-260° C. ¹H NMR (300 MHz, d⁶-DMSO) δ2.80-2.85 (m, 2H), 3.40-3.46 (m, 2H), 7.22-7.38 (m, 2H), 7.51 (d, 1H,J=9.0 Hz), 8.28 (d, 1H, J=9 Hz), 8.72 (m, 1H), 12.41 (s, 1H); ¹³C NMR(74.47 MHz, d⁶-DMSO) δ 36.7, 44.2, 112.7, 113.8, 122.8, 122.9, 125.0,126.2, 134.7, 135.7, 162.5, 195.3; IR: (KBr-pellet) 3209, 1664, 1630,1523, 1437, 1408 cm⁻¹; LRMS (EI): M⁺=214.3; HRMS m/e calcd forC₁₂H₁₀N₂O₂ (M) 214.0742, found 214.0740.

Synthesis of5-(5-oxo-2-phenyl-oxazol-4-ylidene)-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 10, was as follows. A solution of TiCl₄ (132 μL, 1.2 mmol ) inCH₂Cl₂ (1.2 mL) and the mixture was added into THF (3 mL) at 0° C.Compound 8 (65 mg, 0.3 mmol) and 2-phenylazlactone (97 mg, 0.6 mmol)were subsequently added to this mixture. The reaction mixture wasstirred at 0° C. for an additional 2 hours, after which it was allowedto stir overnight at room temperature. NH₄Cl (8 mL, sat. solution inwater) was added and the mixture was stirred for 10 minutes. The mixturewas subsequently extracted with ethyl acetate (3 times), the extractscombined, dried with anhydrous Na₂SO₄, filtered, concentrated and theresidue was purified with column chromatography on silica gel (acetone)to yield compound 10 (59 mg, 55% ) as a foamy yellow solid. Mp: 247-250°C. ¹H NMR (300 MHz, d⁶-DMSO) δ 3.36-3.47 (m, 4H), 7.14 (t, 1H, J=8.1Hz), 7.30 (t, 1H, J=9.0 Hz), 7.49-7.58 (m, 4H), 7.81-7.87 (m, 3H),8.45-8.56 (m, 1H), 12.11(s, 1H); ¹³C NMR (74.47 MHz, d⁶-DMSO) δ 37.7,38.6, 112.9, 115.3, 121.0, 125.1, 125.3, 126.5, 127.4, 129.0, 129.8,133.2, 134.0, 136.9, 146.3, 158.4, 165.0, 166.1; IR: (KBr-pellet) 3358,3179, 1749, 1653, 1633, 1448 cm⁻¹; HRMS m/e calcd for C₂₁H₁₅N₃O₃ (M)357.1113, found 357.1106.

Synthesis of5-(2-amino-5-oxo-1,5-dihydro-imidazol-4-ylidene)-3,4,5,10-tetrahydro-2H-azepino[3,4-b]indol-1-one,compound 2, was as follows. LiH (16 mg, 2 mmol) was dissolved in ethanol(60 mL) and to this solution was added compound 10 (150 mg, 0.4 mmol)and S-benzylisothiouronium chloride (405 mg, 2 mmol). The reaction wasrefluxed 48 hours after which the reaction was cooled and the solventwas evaporated in vacuo. Ethanol (20 mL) added and subsequentlyevaporated 3 times. Ethanol (5 mL) was added again and the reaction wasrefluxed for 3 hours. The solvent was evaporated off in vacuum, 1NHCl_((aq)) (15 mL) was added and the mixture was extracted withn-butanol (3×15 mL), the extracts were washed with brine (3×10 mL),dried, concentrated and the product was purified by columnchromatography on silica gel (CH₂Cl₂:MeOH:NH₃.H₂O, 3:1:0.1) to yieldcompound 2 (35 mg, 30%) as light yellow solid, Mp:>260° C.; ¹H NMR (300MHz, d⁶-DMSO) δ 3.20-3.40 (br., 4H), 7.16 (t, 1H, J=12.0 Hz), 7.29 (t,1H, J=12.0 Hz), 7.52 (m, 2H), 8.30-8.50 (m, 2H), 9.05-9.25 (br., 1H),10.30-10.40 (m, 1H), 12.48 (s, 1H); ¹³C NMR: (124.1 MHz), d⁶-DMSO ) δ36.5, 39.2, 112.7, 113.5, 121.9, 122.3, 122.8, 124.5, 125.0, 128.6,132.8, 137.0, 154.6, 163.4, 165.5; IR: (KBr-pellet) 3294, 1620, 1475,1251 cm⁻¹; LRMS (EI): 295.1 (M) ; HRMS (FAB) calcd for C₁₅H₁₄N₅O₂ (M+H)296.1148, found 296.1144.

EXAMPLE 2

This example shows that compound 2, like hymeniadisine 1, exhibits asignificant dose response inhibition of IL-2 production when measured ina competitive enzyme immunoassay (EIA) for IL-2 expression.

Human Jurkat leukemia T-cells (clone E6-1; ATCC No. TIB-152, AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.) aregrown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.) supplemented with10% fetal bovine serum, penicillin (614 ηg/mL), streptomycin (10 μg/mL)and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. To each well of a flatbottomed 96 well culture plate 0.2 mL 1×10⁶ Jurkat E6-1 cells/mL wereadded. Each sample was then treated in duplicate with the compounds inDMSO at either 10 μM, 1 μM, 0.1 μM or 10 ηM and allowed to incubate forthirty minutes at 37° C., 5% CO₂. Cell free supernatant fractions werecollected from stimulated cultures incubated for 24 hr at 37° C., 5%CO₂. Cultures were stimulated with phytohemagglutinin (PHA,Sigma-Aldrich, St. Louis, Mo.) at 1 μg/mL; and phorbol myristate acetate(PMA, Sigma-Aldrich, St. Louis, Mo.) at 50 ng/mL. The concentration ofIL-2 in each sample was then measured using a competitive enzymeimmunoassay (Neogen Corporation, Lansing, Mich.) according to themanufacturer's protocol. Known IL-2 concentrations were plotted and fita 4 parameter logistic curve. Unknown concentrations were thenextrapolated from the standard curve.

Hymenialdisine 1 exhibited significant inhibition of IL-2 productionwith an IC₅₀ value of 2.4 μM (Table 1 and FIG. 2B). Treatment of theJurkat cells with compound 2 at concentrations ranging from 0.1 μM to 10μM exhibited also a significant dose response inhibition of IL-2production (FIG. 2C). Compound 2 exhibited an inhibition of IL-2production with an IC₅₀ value of 3.5 μM.

Interestingly, blockage of the N-indole position with a methyl group,compound 3, indicated no significant inhibition of IL-2 production up tothe concentration tested (FIG. 2D, measured up to 10 μM). This furthersupports the significance of the pyrrolic —H-moiety in potentialhydrogen bonding interactions (Meijer et al., Chem. Biol. 7: 51-63(2000)). FIG. 2A shows the effect of PDTC on the cells.

TABLE 1 Inhibition of IL-2 production measured by EIA for PDTC,hymenialdisine 1 and compounds 2 and 3 in PMA/PHA-activated Jurkat Tcells. Inhibition of IL-2 Compounds Production IC₅₀, (μM)^(a) PDTC(control) 5.123 (±0.407) Hymenialdisine 1 2.411 (±0.710) Compound 23.547 (±0.092) Compound 3 >10 ^(a)Values are means of two experiments,standard deviation is given in parentheses.

EXAMPLE 3

This example shows that compound 2, like hymeniadisine 1, exhibits asignificant inhibition of NF-κB-DNA binding when measured in an EMSAassay for NF-κB-DNA binding in which the nuclear extracts of PMA/PHAactivated Jurkat cells were examined for inhibition of DNA binding ofNF-κB by compounds 1-3 using gel electrophoresis (EMSA).

Human Jurkat leukemia T-cells (clone E6-1; ATCC No. TIB-152, AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.) aregrown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.) supplemented with10% fetal bovine serum, penicillin (614 ηg/mL), streptomycin (10 μg/mL)and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. The Jurkat cells (1×10⁶cells/mL) are subsequently treated with various concentrations of thecompounds in DMSO for 30 min. at 37° C. and 5% CO₂ followed by PMA (50ng/mL) and PHA (1 μM/mL) stimulation for an additional 30 minutes. Thecells were harvested by centrifugation, washed in ice cold PBS and thenuclear extracts were prepared as previously described (Dignam et al.,Nucl. Acids Res. 11: 1475-1489 (1983)). The protein concentration of theextracts was determined according to the Method of Bradford (1976) withBioRad reagents.

Nuclear extracts were incubated for 20 minutes at room temperature witha double stranded Cy3 labeled NF-κB consensus oligonucleotide,5′-AGTTGAGGGGACTTTC CCAGGC-3′ (SEQ ID NO:1). The binding mixture (25 μL)contained 10 mM HEPES-NaOH pH 7.9, 4 mM Tris-HCL, pH 7.9, 6.0 mM KCl, 1mM EDTA, 1 mM DTT, 10% glycerol, 0.3 mg/mL bovine serum albumin, and 1μg of poly (dI:dC). The binding mixtures (10 μg of nuclear extractprotein) were incubated for 20 minutes at room temperature with 0.16pmol of Cy3 labeled oligonucleotide. The mixture was loaded on a 4%polyacrylamide gel prepared in 1× TRIS borate/EDTA buffer and waselectrophoresed at 200 V for 20 minutes. After electrophoresis the gelwas analyzed using a phosphorimager (BIORAD FX PLUS, BioradLaboratories, Inc., Hercules, Calif.) for detection of NF-κB-DNAbinding.

The results are shown in FIG. 3 and Table 2. Control lanes included lane1, in which the NF-κB p65 antibody was used to confirm the NF-κB-DNAcomplex (FIG. 3). Addition of the antibody to the nuclear extract ofactivated Jurkat cells resulted in a supershift indicated in FIG. 3(lane 1). The DNA-NF-κB complex is illustrated in lane 2. Lane 3includes the DNA binding of a non stimulated cell extract, which asanticipated resulted in no significant band shift (lane 3). Lanes 4 and5, were treated with 5 μM and 1 μM PDTC, respectively as a positivecontrol for NF-κB-DNA binding inhibition.

Inhibition of NF-κB-DNA binding was found to be 51% and 30% for 5 μM and1 μM PDTC, respectively (lanes 4 and 5) as compared to the activatedcontrol (lane 2). Hymenialdisine at 5 μM (46% inhibition) and 1 μMconcentration (3% inhibition, lanes 6 and 7, respectively) indicatedsignificant inhibition of DNA binding as reported earlier (Roshak etal., J. Pharmacol. Exp. Ther. 283: 955-961 (1997)). Compound 2,demonstrated similar inhibition of NF-κB-DNA binding as the naturalproduct hymenialdisine with 49% and 22% inhibition at 5 μM and 1 μM,respectively (lanes 8 and 9). Compound 3 did not appear to substantiallyinhibit DNA binding as measured up to concentrations of 5 μM (lanes 10and 11). In fact, a small increase in binding was observed as comparedto the activated control. A summary of the percent inhibition relativeto the activated control is listed in Table 3. Similar to the studiesreported on hymenialdisine (Roshak et al., J. Pharmacol. Exp. Ther. 283:955-961 (1997)), no significant inhibition of DNA binding was observedwhen compounds 1-3 were tested for inhibition of DNA binding with thetranscription factor AP-1.

TABLE 2 Inhibition of NF-κB-DNA binding measured by EMSA for PDTC,hymenialdisine 1 and compounds 2 and 3 in PMA- activated Jurkat cells.Inhibition of DNA-binding (relative to Count per activated mm² NF-κBCompounds control) bands Activated cells — 105.55 (control) 5.0 μM PDTC(control) 51% 51.68 1.0 μM PDTC (control) 30% 73.48 5.0 μMHymenialdisine 1 46% 57.06 1.0 μM Hymenialdisine 1 3% 102.14 5.0 μMCompound 2 49% 53.40 1.0 μM Compound 2 22% 82.38 5.0 μM Compound 3 0%121.76 1.0 μM Compound 3 0% 118.82

EXAMPLE 4

This example shows that compound 2, like hymenialdisine 1, exhibits asignificant dose response inhibition of TNF-α when measured in acompetitive enzyme immunoassay (EIA) for TNF-α expression. The assaymeasured the ability of the compounds to inhibit TNF-α production in LPSstimulated THP-1 cells (Aikawa et al., Inflamm. Res. 51V 188-194(2002)).

THP-1 cells are grown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.)supplemented with 5% fetal bovine serum, penicillin (614 ηg/mL),streptomycin (10 μg/mL) and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. Toeach well of a flat bottomed 96 well culture plate 0.2 mL 1×10⁶ THP-1cells/mL were added. Each sample was then treated in duplicate with thecompounds in DMSO at either 10 μM, 1 μM, 0.1 μM or 10 ηM and allowed toincubate for thirty minutes at 37° C., 5% CO₂. Cell free supernatantfractions were collected from stimulated cultures incubated for 3 hr at37° C., 5% CO₂. Cultures were stimulated with LPS (Sigma-Aldrich, St.Louis, Mo.) at 1 μg/mL. The concentration of TNF-α in each sample wasthen measured using a competitive enzyme immunoassay (NeogenCorporation, Lansing, Mich.) according to the manufacturer's protocol.To make a standard curve, known TNF-α concentrations were plotted andfitted to a 4 parameter logistic curve. Unknown concentrations were thenextrapolated from the standard curve.

Treatment of THP-1 cells with PDTC and compounds 1-3, 30 minutes priorto LPS activation, resulted in a significant inhibition of TNF-αproduction after a 3 hour incubation period (Table 3 and FIG. 5).Hymenialdisine 1 was found to be a potent inhibitor of TNF-α productionwith an IC₅₀ value of 1.4 μM. Compound 2 was also found to inhibit TNF-αproduction, albeit less potent than hymenialdisine (IC₅₀=8.2 μM). Themethylated indoloazepine 3 did not have any significant activity at theconcentrations tested (tested up to 10 μM, Table 3).

TABLE 3 Inhibition of TNF-α production measured by EIA for PDTC,hymenialdisine 1 and compounds 2 and 3 in PMA/PHA-activated THP-1 cells.Inhibition of TNF-α Compounds Production IC₅₀, (μM)^(a) PDTC (control)6.109 (±0.529) Hymenialdisine 1 1.357 (±0.253) Compound 2 8.161 (±0.313)Compound 3 >10 ^(a)Values are means of two experiments, standarddeviation is given in parentheses.

EXAMPLE 5

Hymenialdisine 1 and compounds 2 and 3 were tested for theircytotoxicity and were found to exhibit significant inhibition of cellgrowth. Cells were treated at different concentrations ranging from 10ηM to 5 μM concentrations over a 48 hour time period.

CEM cells (CCRF-CEM; ATCC No. CCL-119, American Type Culture Collection,Manassas, Va.) were grown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.)supplemented with 10% Fetal Bovine Serum, penicillin (614 ηg/mL),streptomycin (10 μg/mL) and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. DMSOwas used as the vector for all drugs and added in the controlexperiments. Cell cultures were then treated with 10 μM, 1 μM, 0.1 μM or10 ηM of the drug in duplicate and allowed to incubate at 37° C., 5%CO₂. Cells were stained with 4% trypan blue solution in PBS, and countedunder the microscope (Thomas Scientific, Fisher Scientific) induplicate. This was repeated for 0 h, 2 h, 6 h, 12 h, 24 h, and 48 h.The data points obtained were then plotted and a point-to-point curvewas drawn to determine the effect of the drug on the cells. FIG. 6Acompares the cytotoxicity of hymenialdisine to compounds 2 and 3 andFIG. 6B shows the growth patterns in CEM cells at differentconcentrations of compound 2. The figures show that compound 3 was lesscytotoxic than compound 2 or hymenialdisine.

Hymenialdisine and compounds 2 indicated similar inhibition of cellgrowth with GI₅₀ of 1.61 μM and 1.73 μM respectively, whereas compound 3indicated weaker inhibition of cell growth (GI₅₀=14.3 μM). Theinhibition of cell growth with compounds 1-3 could therefore account forsome of the inhibition of IL-2 production seen in the Jurkat cells. IL-2production was measured 24 hours after PMA stimulation. However,inhibition of TNF-α production was measured within 3 hours after LPSstimulation and no significant inhibition of cell growth was apparent atthis time interval.

TABLE 4 Inhibition of cell growth by hymenialdisine 1 and compounds 2and 3 in CEM leukemia cells. Inhibition of cell growth Compounds GI₅₀,(μM)^(a) PDTC (control) N/T Hymenialdisine 1 1.61 (±0.062) Compound 21.73 (±0.088) Compound 3 14.3 (±2.41)  ^(a)Values are means of twoexperiments, standard deviation is given in parentheses.

EXAMPLE 6

Compounds 2 and 3 were tested for inhibition of the cyclin dependentkinases CDK1 and GSK-3β using the method of Meijer et al., Chem. Biol.7: 51-63 (2000) for showing inhibition of the kinases by hymenialdisine.The results are shown in FIGS. 7A-C and summarized in Table 5.

TABLE 5 Inhibition of CDK1 and GSK-3 by hymenialdisine 1 and compounds 2and 3. Inhibition Inhibition of CDK1 of GSK-3β Compounds IC₅₀, (μM)IC₅₀, (μM) Hymenialdisine 1 0.06 0.045 Compound 2 0.4 0.15 Compound3 >10 >10Like hymenialdisine, compound 2 inhibited CDK1 (IC₅₀=0.4 μM) and GSK-3β(IC₅₀=150 ηM). Inhibition by compound 3 was less pronounced. The resultsare consistent with the reported X-ray crystal structure ofhymenialdisine where the pyrrolic hydrogen was found to be involved in ahydrogen bonding interaction in the ATP-binding pocket (Meijer et al.,Chem. Biol. 7: 51-63 (2000)).

Table 6 illustrates the comparison between the in vitro activity ofindoloazepine 2, hymenialdisine and debromohymenialdisine. Compound 2exhibits very potent inhibition of CHK2 activity in the low nanomolarrange. Interestingly, unlike the natural product hymenialdisine anddebromohymenialdisine, compound 2 exhibits also very potent selectivityfor the checkpoint kinase CHK2.

TABLE 6 Table 6. IC₅₀ values for kinase inhibition by compound 2,hymenialdisine and debromohymenialdisine. IC₅₀ (nM) hymenialdisineKinase compound 2 10, 12 debromohymenialdisine CK1δ(h) 1,352   35 NACK2(h) >10,000 7,000 NA MEK1(h) 89    6¹³    824¹³ PKCα(h) 2,539   700NA PKCβII(h) 3,381 1,200 NA CHK1 237 NA 3,000⁷ CHK2 8 NA 3,500⁷ NA:value not available

EXAMPLE 7

The potent inhibitory activity of the indoloazepines against checkpointkinases Chk1 and Chk2 was determined. In vitro kinase Assay: Compound 2was tested in vitro against the kinases indicated by Upstate, UK using aKinase Profiler Assay according to the manufacturer's protocol. Briefly,in a final volume of 25 μl, the kinase was incubated with the desiredbuffer and the required polypeptide substrate, in presence of 10 mMmagnesium acetate and γ-³³P-ATP (10 μM). After incubation for 40 minutesat room temperature, the reaction was stopped by the addition of 3%H₃PO₄ (5 μl). A 10 μl of the reaction was then spotted on a P30filtermat and washed 3× in 75 mM H₃PO₄ and finally in methanol. Sampleswere then dried and signals counted on a scintillation counter. Theindoloazepine exhibits very potent inhibition of Chk2 activity in thelow nanomolar range (IC₅₀=8 nanoMolar). Unlike the natural producthymenialdisine and debromohymenialdisine, the indoloazepine exhibitsalso very potent selectivity for the checkpoint kinase Chk2. Thesekinase inhibition studies suggest that analogs of hymenialdisine mayimprove its overall kinase profile and significantly affect its kinaseselectivity. The results are shown in FIGS. 8 and 9 for CHK1 and CHK2.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1. A compound of the formula

and amine salts thereof, wherein R₁, R₂, and R₃ are each selected fromthe group consisting of hydrogen, alkyl containing 1 to 6 carbon atoms,halo, aryl, hydroxyl, amino, SH, carboxyalkyl, alkoxy and carboxyamidomoieties.
 2. The compound of claim 1 wherein R₁, R₂, and R₃ are eachhydrogen.